Identification of phenylcarbamoylazinane-1,3,4-oxadiazole amides as lipoxygenase inhibitors with expression analysis and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2021.105243

In search for new anti-inflammatory agents that inhibit the enzymes of arachidonic acid pathway as the drug targets, the present article describes the screening of 1,3,4-oxadiazole analogues against lipoxygenase (LOX) enzyme. The work is based on the synthesis of new N-alkyl/aralky/aryl derivatives (6a-o) of 2-(4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,3,4-oxadiazol-3-ylthio)acetamide which were obtained by the reaction of 1,3,4-oxadiazole (3) with various electrophiles (5a-o), in KOH. The synthesized analogues showed potent to moderate inhibitory activity against the soybean 15-LOX enzyme; especially 6g, 6b, 6a and 6l displayed the potent inhibitory potential with IC₅₀ values 7.15 ± 0.26, 9.32 ± 0.42, 15.83 ± 0.45 & 18.37 ± 0.53 μM, respectively, while excellent to moderate inhibitory profiles with IC₅₀ values in the range of 26.13–98.21 μM were observed from the compounds 6k, 6m, 6j, 6o, 6h, 6f, 6n and 6c. Most of the active compounds exhibited considerable cell viability against blood mononuclear cells (MNCs) at 0.25 mM by MTT assay except 6f, 6h, 6k and 6m which showed around 50% cell viability. Flow cytometry studies of the selected compounds 6a, 6j and 6n revealed that these caused 79.5–88.51% early apoptotic changes in MNCs compared with 4.26% for control quercetin at their respective IC₅₀ values. The relative expression of 5-LOX gene was monitored in MNCs after treatment with these three molecules and all down-regulated the enzyme activity. In silico ADME and molecular docking studies further supported these studies of oxadiazole derivatives and considered it as potential ‘lead’ compounds in drug discovery and development.

Full text of DOI:10.1016/j.bioorg.2021.105243

Bioorganic Chemistry 115 (2021) 105243
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of phenylcarbamoylazinane-1,3,4-oxadiazole amides as
lipoxygenase inhibitors with expression analysis and in silico studies
,
Bushra Bashira, Wardah Shahida, Muhammad Ashrafa *, Muhammad Saleema,
Aziz-ur-Rehmanb, Saima Muzaffara, Muhammad Imrana, Hira Amjadb, Keshab Bhattaraic,
,
*
Naheed Riaza
a Institute of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
b Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan
c Department of Pharmaceutical Biology, Auf der Morgenstelle 8, 72076, University of Tuebingen, Tuebingen, Germany
A R T I C L E I N F O
A B S T R A C T
Keyword:
In search for new anti-inflammatory agents that inhibit the enzymes of arachidonic acid pathway as the drug
targets, the present article describes the screening of 1,3,4-oxadiazole analogues against lipoxygenase (LOX)
enzyme. The work is based on the synthesis of new N-alkyl/aralky/aryl derivatives (6a-o) of 2-(4-phenyl-5-(1-
phenylcarbamoylpiperidine)-4H-1,3,4-oxadiazol-3-ylthio)acetamide which were obtained by the reaction of
1,3,4-oxadiazole (3) with various electrophiles (5a-o), in KOH. The synthesized analogues showed potent to
moderate inhibitory activity against the soybean 15-LOX enzyme; especially 6g, 6b, 6a and 6l displayed the
potent inhibitory potential with IC50 values 7.15 ± 0.26, 9.32 ± 0.42, 15.83 ± 0.45 & 18.37 ± 0.53 µM,
respectively, while excellent to moderate inhibitory profiles with IC50 values in the range of 26.13–98.21 µM
were observed from the compounds 6k, 6m, 6j, 6o, 6h, 6f, 6n and 6c. Most of the active compounds exhibited
considerable cell viability against blood mononuclear cells (MNCs) at 0.25 mM by MTT assay except 6f, 6h, 6k
and 6m which showed around 50% cell viability. Flow cytometry studies of the selected compounds 6a, 6j and 6n
revealed that these caused 79.5–88.51% early apoptotic changes in MNCs compared with 4.26% for control
quercetin at their respective IC50 values. The relative expression of 5-LOX gene was monitored in MNCs after
treatment with these three molecules and all down-regulated the enzyme activity. In silico ADME and molecular
docking studies further supported these studies of oxadiazole derivatives and considered it as potential ‘lead’
compounds in drug discovery and development.
Lipoxygenase inhibition
In silico studies
Expression analysis
Azinane-oxadiazoles
Synthesis
MTT assay
ADME studies
1. Introduction
hydroperoxides which are further converted into biologically active
molecules involved in inflammation. COX-I (EC 1.14.99.1) is implicated
The ever increasing number of many sinister diseases towards
available drugs applications like the COVID-19, demand the discovery of
new multifunctional drug molecules [1]. Inflammation is a primary
immune reaction in response to all tissue injuries and viral or bacterial
infections albeit is involved in regulation of tissue homeostasis. There-
fore, it is linked with many diseases like asthma, osteoarthritis, rheu-
matoid arthritis, carcinoma, diabetes mellitus, Crohn’s disease,
Alzheimer’s disease and multiple sclerosis [2]. In this regard, cyclo-
oxygenase (COX) and lipoxygenase (LOX) pathways play central role in
the development and progression of acute to chronic inflammatory
response associated with the involvement of other mediators. COX and
LOX enzymes catalyze arachidonic acid and linoleic acid to
in platelet aggregation and renal homeostasis. COX-II (EC 1.14.99.2) is
inducible enzyme that catalyzes the synthesis of prostaglandin G2,
thromboxanes, prostacyclin and other molecules that mediate pain,
heat, redness, swelling and fever [3] (Scheme 1).
In LOX pathway, lipoxygenases (LOXs, EC 1.13.11.12) are involved
and constitute the family of enzymes present across multiple species
from plants to mammals [4]. The important plants LOXs are 5-LOX and
15-LOX whilst important mammalian LOXs are 5-LOX, 8-LOX, 12-LOX,
and 15-LOX. LOXs from plants and mammals have the highest level of
sequence identity in the area of catalytic domain containing non-heme
iron atom. Literature shows that soybean 15-LOX inhibitors are also
good inhibitors of human 5-LOX or sometime better than the soybean
* Corresponding authors.
E-mail addresses: dr.m.ashraf@gmail.com (M. Ashraf), nrch322@iub.edu.pk (N. Riaz).
https://doi.org/10.1016/j.bioorg.2021.105243
Received 22 February 2021; Received in revised form 16 July 2021; Accepted 2 August 2021
Available online 4 August 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
15-LOX. The biological properties of human LOXs are widely studied
because of the involvement in several inflammatory diseases including
cancer [5-7].
and in some tissues trioxilin C3 is also formed. 12/15-LOX isoforms can
also produce 15-HPETEs. 15-LOX and 5-LOX in the sequential action
produce trihydroxytetraenes (lipoxins) [8,9] (summary is given in
Scheme 1). Most of these active metabolites are implicated in various
disorders including sinusitis, cataracts, pain, heart disease, stroke,
asthma, arthritis, colitis etc [2,8,9]. Therefore, the inhibition of COX and
LOX enzymes by small molecules provide an opportunity in search for
‘lead’ compounds for the treatment of inflammatory disorders including
some types of cancer that can give some relief to patients. Zileuton (N-
When arachidonic acid is metabolized, all of the different LOX iso-
forms generate respective hydroperoxides (HPETEs) as the primary
products that are rapidly converted into their corresponding hydroxides
(HETEs). These HPETEs serve as precursors for the generation of other
classes of secondary lipid mediators. For example, 12/15-LOX generates
12-HPETE that could be converted to hepoxilin A3 and hepoxilin B3,
Scheme 1. Overview of the arachidonic acid metabolism. Key details are given in the text.
2

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea) is the only 5-LOX inhibi-
tor that has been approved and is marketed for the treatment of asthma
[10].
To further advance our ongoing research for the development of
unique LOX inhibitors and distinctive biological potential of oxadiazole
family encouraged us to design structural analogues of 1,3,4-oxadiazole
for the hope of finding new inhibitors with better activity. Herein, we
have synthesized a series of new N-alkyl/aralky/aryl oxadiazole de-
rivatives (6a-o) and investigated them for their LOX inhibitory potential
followed by toxicity studies, in silico studies and expression profiles on
human 5-LOX in mononuclear cells in search for anti-inflammatory
‘lead’ molecules.
Heterocyclic compounds especially those containing nitrogen atoms
are of great importance in life sciences and represent 85% of all bio-
logically active compounds and therefore are prime targets for synthetic
medicinal chemists and biologists [11,12]. It has been reported that 60%
of small molecule drugs are comprised of nitrogen-based drugs which is
also because of the fact these molecules bind DNA with hydrogen
bonding and show anticancer and related activities [13,14]. Heterocy-
cles are amongst the most important organic compounds that are
commonly found in the molecules of interest often used for medicinal
purposes like the multiple heteroatom containing heterocycles are of
great importance to life. We are interested in heterocyclic compounds,
especially those containing nitrogen and oxygen atom due to carry of
their diverse medicinal applications [15,16].
2. Results and discussion
2.1. Chemistry
The objective of the present study was to synthesize new
phenylcarbamoylpiperidine-1,3,4-oxadiazole amides (6a-o) containing
a collection of substituents and to explore them for their in vitro enzyme
inhibitory potential against 15-LOX supported by cytotoxicity and
expression analysis data on human MNCs. Synthesis of the intermediate
and the target compounds was finalized according to the protocol as
shown in Scheme 2. The synthesis was started with isonepacotate (a),
which on rubbing with phenylisocyanate and further treatment with
hydrazine hydrate resulted into hydrazide 2. The 1H NMR spectrum of 2
displayed two singlets at δ 4.04 (1H, –NH) and 8.93 (2H, –NH2) which
are characteristics of –NHNH2 moiety. Hydrazide (2) refluxed with CS2
followed by KOH to get intramolecular cyclized product 5–1-(1-phe-
nylcarbamoylpiperidine)-4H-1,3,4-oxadiazole-2-thiol (3). The N-alkyl/
aralkyl/aryl-2-bromoacetamides (5a-o), the second precursors of target
compounds were synthesized by stirring alkyl/aralkyl/aryl amines (4a-
o; Table 1) with 2-bromoacetyl bromide in a basic medium (pH 9–10).
The final step was the combination of compound 3 separately with the
precursors 5a-o to get target compounds 6a-o, respectively. This task
was performed according to the procedures given in the experimental
section.
The chemistry of heterocyclic compounds peculiarly 1,3,4-oxadia-
zoles derivatives started in the last few decades. The competency of
1,3,4-oxadiazoles endures numerous chemical reactions with immense
biological applications. The five membered ring derived by substituting
two pyridine nitrogen atoms in furan comprises 1,3,4-oxadiazole, a
developing step for variety of heterocycles [17]. Nitrogen has different
position in oxadiazole ring, that’s why four isomers of oxadiazole are
1,3,4-oxadiazole, 1,2,4-oxadiazole, 1,2,3-oxadiazole and 1,2,5-oxadia-
zole exist. Yet, 1,2,4-oxadiazole and 1,3,4-oxadiazole are familiar and
intensively studied by virtue of their biological potency [18] which
–
arises mainly due to 1,3,4-oxadiazoles having toxophoric –N C–O–
–
correlation. They also act as bioisoesters of ester and amide groups along
with valuable rise in biological action due to hydrogen bonding with
various receptors [19] and the pathological conditions might be
controlled by 1,3,4-oxadiazoles [20]. The 2,5-substituted oxadiazoles
have been reported to possess anticonvulsant, antibacterial [21], anti-
microbial [22], anti-tubercular, insecticidal [23], anti-mycobacterial
[24], anti-inflammatory [25], anticancer [26], analgesic and antiepi-
leptic activities [27]. There are some recommended agents having the
oxadiazole ring like furamizole that are used as antimicrobial and anti-
retroviral, raltegravir and nesapidill are antihypertensive agents [28].
The compounds containing oxadiazoles core are found inhibitors of
The compound 6a was synthesized as a white amorphous powder.
The functional group was identified by infrared (IR) spectrum which
showed the absorption bands at 3362, 3030–, 2933–, 168–0, 1659,
1615–1550, 1250 cmꢀ 1 for N H, Ar H, C H, C O, C C, C N, C
– – –
–
N
– – –
functionalities, respectively. Its 1H NMR spectrum displayed two sets of
triplet and quatret at δ 1.13 (3H, t, J = 7.2 Hz, H-2′′′′), 1.27 (3H, t, J =
7.2 Hz, H-2′′′′′), and 3.41 (2H, q, J = 7.2 Hz, H-1ʹʹʹʹ), 3.49 (2H, q, J = 7.2
Hz, H-1′′′′′), respectively, designated the presence of two N-ethyl groups
in the molecule. The signals that indicate the existence of azinane ring
thymidylate synthase [29],
α
-glucosidase [30], aldose reductase [31],
[32], and carbonic anhydrase I, II, IX,
human DNA topoisomerase II
α
and XII [33]. Previously, we reported α-glucosidase inhibitory p-chlo-
rinated phenyl oxadiazoles by using five-step novel synthetic approach
with promising inhibitory results [34].
Scheme 2. Protocol for the synthesis of oxadiazole amides (6a-o) from isonepacotate.
3

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
Table 1
Alkyl/aralkyl/aryl substituted amines.
Compd
R
Compd
f
R
Compd
k
R
a
b
g
l
c
h
i
m
n
d
e
j
o
coupled to 1,3,4-oxadiazole were appeared at δ 1.81 (4H, m, H-3ʹ,5ʹ),
3.24 (1H, m, H-4ʹ) and 4.16 (4H, m, H-2ʹ,6ʹ). A singlet for a methylene
linked amide with oxadiazole was appeared at δ 4.36 (2H, s, H-2′′′) while
the aromatic region of the same spectrum displayed two triplets at δ 7.01
(1H, J = 7.7 Hz, H-4ʹʹ), 7.26 (2H, t, J = 7.7 Hz, H-3ʹʹ,5ʹʹ) and a doublet at δ
7.35 (2H, d, J = 7.7 Hz, H-2ʹʹ,6ʹʹ) referred to phenyl group. The 13C NMR
spectra (both BB & DEPT) showed total sixteen carbon signals for 20
carbons which corroborated the presence of two methyl, five methylene,
four methine and five quaternary carbon atoms. The most downfield
signals at δ 171.4, 167.6, 165.8 and 157.8 were attributed to two amide
carbonyls and two quaternary carbons of oxadiazole. The signals at δ
140.9, 129.6, 124.1 and 122.2 marked the presence of a phenyl ring
attached to nitrogen atom. The signals for nitrogen attached ethyl
groups appeared at δ 43.9, 42.2, 14.3, & 13.1. The signals for the azinane
ring were appeared at δ 44.5, 34.5 and 30.1. The molecular formula
C20H27N5O3S was inferred by using the data of HR-EI-MS which showed
Fig. 1. Plausible EI-MS fragmentation pattern of compound 6a.
4

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
Table 2
15-LOX inhibitory and cytotoxicity profiles of compounds 6a-o (Mean ± SEM, n = 3).
Comp
Soybean 15-LOX assay
MTT assay
Inhibition (%) at 0.25 mM
IC50 (µM)
Cell viability (%) at 0.25 mM
6a
96.32 ± 1.15
94.17 ± 1.86
67.48 ± 1.59
7.54 ± 1.43
21.64 ± 1.65
76.31 ± 1.87
92.42 ± 1.54
76.13 ± 1.65
74.65 ± 1.78
63.57 ± 1.82
97.54 ± 1.74
93.65 ± 1.83
58.43 ± 1.92
81.35 ± 1.76
91.47 ± 1.65
92.63 ± 1.24
93.79 ± 1.27
–
15.83 ± 0.45
9.32 ± 0.42
98.21 ± 0.38
>250
83.21 ± 1.4
72.55 ± 1.6
84.39 ± 1.5
78.64 ± 1.4
71.85 ± 1.6
52.78 ± 1.7
73.69 ± 1.5
49.57 ± 1.4
76.92 ± 1.7
72.76 ± 1.5
51.68 ± 1.6
98.57 ± 1.6
53.23 ± 1.8
71.25 ± 1.9
75.57 ± 1.8
Not tested
6b
6c
6d
6e
>250
6f
52.94 ± 0.17
7.15 ± 0.26
52.26 ± 0.35
57.45 ± 0.42
46.72 ± 0.54
26.13 ± 0.37
18.37 ± 0.53
42.59 ± 0.49
78.25 ± 0.35
47.56 ± 0.52
4.86 ± 0.14
2.24 ± 0.13
–
6g
6h
6i
6j
6k
6l
6m
6n
6o
Quercetin
Baicalein
Cyclophosphamide
Cisplatin
Curcumin
Not tested
56.5 ± 1.9
51.7 ± 1.8
73.9 ± 1.9
–
–
–
–
the molecular ion [M]+ peak at m/z 417.18346 (detailed EI-MS frag-
mentation pattern is shown in Fig. 1). The spectral data for the
remaining compounds 6b-o of the series is presented in the experimental
section.
said enzyme (Fig. 2). Despite of the fact that the activity is a collective
effect of the whole molecule, so for a minimum SAR have been accepted
through analyzing the effect of various alkyl/aralkyl/aryl groups on
inhibitory capability. The compounds 6 g and 6b expressed potent LOX
inhibitory activities with IC50 values of 7.15 ± 0.26 and 9.32 ± 0.42 µM,
respectively. While evaluating the inhibiting potency of 6b (9.32 ± 0.42
µM) with 6a (15.83 ± 0.45 µM), it is found that the saturated chain of
carbon (n-propyl) interconnected to nitrogen is liable for its extra ac-
tivity correlation to four carbons as diethyl groups that resisted the
binding ability with the enzyme because of its stearic effect. By
analyzing the activity of 6 g (7.15 ± 0.26 µM) and 6 h (52.26 ± 0.35
µM), identical substituents (ethyl group) were present at the ortho and
para positions, respectively, which could be explained by means of
2.2. LOX inhibitory activities and SAR studies
The present study investigated the effect of different substitutions at
1,3,4-oxadiazole species for inhibiting potency of synthesized com-
pounds 6a-o against soybean 15-LOX. In vitro 15-LOX inhibition of
compounds 6a-o was carried out and quercetin (IC50 4.86 ± 0.14 µM)
was used as standard (Table 2). Most of compounds manifested
considerable inhibitions (7.15 ± 0.26 to 98.21 ± 0.38 µM) towards the
Fig. 2. Important 15-LOX inhibitors.
5

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
intensive electron donating effect at ortho position in aromatic ring
compelled for combination of nitrogen with enzyme that become
decrepit in para position.
Table 3
Cytotoxic effects of 6a (IC50 15.8 µM), 6j (IC50 46.7 µM), 6n (IC50 78.2 µM) on
MNCs. Assay was carried out at the IC50 values of the compounds including
controls ibuprofen (IC50 14.0 µM) and quercetin (IC50 4.0 µM).
It is seen that the electron donating substituents enhanced the
inhibiting behavior, however, the consequences indicated the incredibly
distinction in activity under of distance among electron donating
groups, wherein the inhibitory activity becomes lesser as the stearic
hindrance is extended in 6j (IC50 46.72 ± 0.54 µM) and 6i (IC50 57.45 ±
0.42 µM) versus 6l (IC50 18.37 ± 0.53 µM) and 6k (IC50 26.13 ± 0.37
µM). Further, the activity improved by inserting the unsaturation such as
in 6o (IC50 47.56 ± 0.52 µM) which provided the more active sites for
inhibiting the enzyme as correlated to 6c (IC50 98.21 ± 0.38 µM) that
lacked unsaturation. The corresponding inhibitory activity of 6b (IC50
9.32 ± 0.42 µM), 6a (IC50 15.83 ± 0.45 µM) and 6c (IC50 98.21 ± 0.38
Sample
Dead Cells
(Upper
Left)
Live Cells
(Lower
Left)
Early Apoptotic
Cells (Lower
Right)
Late Apoptotic
Cells (Upper
Right)
Control
6a
0.80%
0.00%
0.05%
0.13%
0.90%
3.98%
94.85%
3.68%
3.12%
88.51%
79.52%
84.75%
6.15%
1.23%
7.81%
8.92%
3.41%
27.48%
10.90%
6j
11.50%
11.71%
65.39%
80.82%
6n
Ibuprofen*
Quercetin
4.26%
*
Ibuprofen is a potent inhibitor of LOX enzymes[35].
Negative Control
6a
6j
6n
Ibuprofen
Quercetin
Fig. 3. Cytotoxic effect of 6a, 6j and 6n on MNCs. Typical dot plots showing apoptosis ratio comparing negative control and positive control Ibuprofen and Quercetin
using PI and Annexin V-FITC method. The quadrant 1 represents damaged cells (PI positive and Annexin negative), quadrant 2 represents cells that are in late
apoptosis or already dead (both Annexin and PI positive), quadrant 3 represents viable cells (both Annexin and PI negative) and quadrant 4 represents cells in early
apoptosis (Annexin positive and PI negative).
6

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
cells at single concentration of 0.25 mM. Compounds 6f, 6h, 6k, 6m
exhibited 49.57 ± 1.4% to 53.23 ± 1.8% cell viability under the given
concentration and experimental protocol whilst rest of all the com-
pounds showed cell viability of > 70% on the average. It is pertinent to
say that this data should not be compared with the flow cytometry data
of selected compounds 6a, 6j and 6n wherein assays were carried out at
their respective IC50 values (Table 3).
Table 4
Relative expression of 5-LOX gene after treatment of different potent derivatives
at × 3 times the concentration of the IC50 values of the drugs including positive
controls with MNCs. Details are given in the Supplementary Material.
Sr. #
Comp
MNCs
Total Expression
Negative Control
1
1
6a
0.00167
0.00067
0.00080
0.00033
0.00024
2
6j
2.3. Flow cytometry studies
3
6n
Control
Control
Ibuprofen*
Quercetin
The toxic effects of selected compounds 6a (IC50 15.8 µM), 6j (IC50
46.7 µM), 6n (IC50 78.2 µM) with the lowest, the moderate and the high
IC50 values were selected for flow cytometry analysis including
ibuprofen and quercetin as standards. The assays were carried out on
MNCs using FITC-Annexin V and PI method to facilitate the quantifi-
cation of cell damage (quadrant 1) and the identification of late
apoptotic cells/necrotic cells (quadrant 2), viable cells (quadrant 3) and
early apoptotic cells (quadrant 4). The analysis was carried out at the
concentration of IC50 values (Fig. 3). The total 10,000 events were
observed for the determination of viability and death of MNCs. It was
observed that these molecules showed different effects on cell viability
as compared with the controls. Data shows that compounds 6a, 6j and
6n induced early apoptosis in cells (79.52% to 88.51%) compared with
the controls, ibuprofen (6.15%) and quercetin (4.26%). These com-
pounds exhibited 3.41% to 8.92% late apoptotic changes compared with
the control drugs (10.9% to 27.48%) for quercetin and ibuprofen,
respectively. The percentage of live cells was greater in the control
groups compared with the 6a, 6j and 6n values which exhibited 3.68%
to 11.71% live cells during the assay conditions.
*
Ibuprofen is a potent inhibitor of LOX enzymes [35].
µM) was found decreased (increased IC50 values) wherein nitrogen is
linked to aliphatic chain rather than the presence of saturated ring
system. Subsequently analyzing the SAR within synthesized derivatives,
it may be argued that the positions as well as nature of substituents
linked to phenyl ring have great impact on 15-LOX inhibitory activity.
MTT assay using MNCs was carried out to observe the viability (%) of
Table 5
ADME properties of compounds 6a-o.
Comp
MlogP
S þ
S þ
M. Wt.
M_NO
T_PSA
HBDH
logP
logD
6a
6b
6c
6d
6e
6f
2.293
2.066
2.950
2.723
2.991
2.991
3.202
3.202
3.202
3.202
3.202
3.202
3.202
3.202
2.776
2.204
2.043
3.370
2.514
2.954
3.004
3.329
3.424
3.219
3.286
3.295
3.215
3.304
3.325
2.723
2.204
2.043
3.370
2.514
2.954
3.004
3.329
3.424
3.219
3.285
3.295
3.215
3.304
3.325
2.723
417.533
403.506
457.598
451.550
451.550
451.550
465.577
465.577
465.577
465.577
465.577
465.577
465.577
465.577
437.523
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
91.57
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
100.36
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
The relative expression analysis of 5/15-LOX gene in MNCs was
carried out as mentioned in the Supplementary Material. MNCs were
incubated with the active molecules 6a, 6j and 6n at × 3 times the
concentration of the IC50 values along with the standard ibuprofen and
quercetin (Table 4). Quercetin and ibuprofen demonstrated down-
regulation of 5/15-LOX in MNCs by 0.00024 and 0.00033 ratio
compared to negative control 1. Compounds 6n showed relative
expression of 0.00080 followed by 6j (0.00067). The down-regulation
by 6a was less pronounced by a ratio of 0.00167 as compared to the
6g
6h
6i
6j
6k
6l
6m
6n
6o
Table 6
The docking score, inhibition constant (Ki) & nature of binding interaction for 6a-o with 5-LOX human.
Ligands/
Compds
Binding
affinity
Ki
Nature of binding interactions
(nMol)
–
–
6a
6b
6c
6d
6e
ꢀ 7.7
ꢀ 7.2
ꢀ 8.5
ꢀ 8.8
ꢀ 8.5
2.251
4.424
0.583
0.351
0.583
Val243-Pip.(Alkyl), Glu287-Me(C H), Leu288-Me(alkyl), Ser447-N(H-B), Leu448-Ar(
π
-alkyl), Gln549-C
–
O(H-B).
-anion), Tyr470-C
-anion), Glu287-Me.(C H), Arg438-Cyclohex.(alkyl), Leu448-Ar(π-alkyl), Gln549-C O(H-B).
–
–
O(H-B).
Glu287-Ar(
π
-anion), Arg370-Oxa.(
π
π
π
–
–
–
–
Val243-Pip.(Alkyl), Arg246-Oxa.(
Val243-Pip.(Alkyl), Arg246-Oxa.(
–
–
-anion), Glu287-Benz.(C H), Arg438-Ar(π-alkyl), Leu448-Ar(π-alkyl), Gln549-C O(H-B).
Leu244-Ar(
Oxa.(
Val243-Pip.(Alkyl), Arg246-Oxa.(
π-alkyl),Me(alkyl), Leu288-Me(alkyl), Val361-Me(alkyl), Ile365-Me(alkyl), Ala446-Pip.(alkyl), Ala453-Pip.(Alkyl),
π
-alkyl), Ala455-Ar(
π
-alkyl), Ala456-Oxa.(
π
-alkyl),Pip.(Alkyl),Ar(
π
-alkyl).
–
–
6f
ꢀ 8.9
ꢀ 7.7
ꢀ 7.8
0.297
2.251
1.901
π
-anion), Leu288-C O(H-B),Ar(
π
-alkyl), Arg438-Ar(π-alkyl), Lys441-Me(alkyl), Ser447-Pip.
–
(C H), Leu448-Ar(
Leu244-Me(alkyl), Leu288-Me(alkyl),Ar(
Ala455-Ar( -alkyl), Ala456-Oxa.( -alkyl),Pip.(Alkyl),Ar(
Leu244-Ar( -alkyl),Me(alkyl), Phe286-Me(alkyl), Val361-Me(alkyl), Ala446-Pip.(alkyl), Ala453-Oxa.(
Ala456-Oxa.( -alkyl), Pip.(Alkyl).
Ala453-Ar( -alkyl),Me(C H), Ala456-Oxa.(
Leu244-Ar( -alkyl), Phe286-Me(alkyl), Leu288-Ar(
Pip.(alkyl), Ala453-Oxa.( -alkyl), Ala455-Ar( -alkyl), Ala456-Oxa.(
Val243-Me(alkyl), Leu244-Ar( -alkyl), Arg246-Oxa.( -anion), Cys248-Ar(
Val361-Me(alkyl), Ile365-Me(alkyl).
Val243-Me(alkyl), Leu288-Ar( -alkyl),Pip.(Alkyl), Arg438-Ar(
Leu244-Ar( -alkyl),Me(alkyl), Leu288-Me(alkyl), Val361-Me(alkyl), Ile365-Me(alkyl), Ala446-Pip.(alkyl), Ala453-Oxa.(
π-alkyl).
6g
6h
π
-alkyl), Val361-Ar(
π
-alkyl), Ile365-Me(alkyl), Ala446-Pip.(alkyl), Ala453-Oxa.(
π
π
-alkyl),
-alkyl),
π
π
π
-alkyl).
π
π-alkyl), Ala455-Ar(
π
–
6i
6j
ꢀ 8.3
ꢀ 8.0
0.817
1.356
π
π
π
-alkyl).
-alkyl),Me(alkyl), Val361-Ar(
-alkyl), Pip.(Alkyl).
-alkyl), Glu287-Oxa.(π-anion), Leu288-Me(alkyl),
π
π-alkyl),Me(alkyl), Ile365-Me(alkyl), Ala446-
π
π
π
6k
ꢀ 8.3
0.817
π
π
π
6l
ꢀ 8.8
ꢀ 7.9
0.351
1.606
π
π
-alkyl), Leu448-Me(alkyl), Phe450-Me(alkyl), Ala453-Ar(
π
-alkyl).
6m
π
π
-alkyl),
–
Ser447-Oxa.(C H), Ala455-Ar(
Val29-Me(alkyl), Arg68-Ar( -anion), Arg101-Ar(
-anion), Pip.(Alkyl), Arg138-Ar( -Alkyl).
Arg101-Oxa( -Alkyl),Pip.(Alkyl), Val107-Ar(
),S(π-π), Phe393-S(π-π), Arg401-Ar(π-anion), Phe402-Ar(π
π
-alkyl), Ala456-Oxa.(
π
-alkyl),Pip.(Alkyl),Ar(
π
-alkyl).
-Alkyl), Ile126-Me(alkyl), Lys133-Oxa.
–
–
6n
ꢀ 9.1
ꢀ 8.3
ꢀ 8.0
0.212
0.817
1.356
π
π
-anion),C O(H-B), Val110-Oxa.(π
(
π
π
6o
π
π
-Alkyl), Lys133-Ar(
π
-anion), Glu134-Pip(Alkyl), Thr137-Ar(
-Alkyl).
-Alkyl), Ala453-Ar(
π
-alkyl), Tyr383-Oxa.
(
π
-
π
Quercetin
Val243-Fur.(alkyl), Arg370-fur.(
π
-anion), Leu448-OH(H-B), Ar(
π
π-alkyl), Arg457-Ar(π-anion), Thr545-OH(H-
B).
7

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
Fig. 4. 2D and 3D binding affinity of 6n in 5-LOX human.
controls. These studies clearly show that the synthesis and expression of
5/15-LOX in MNCs is affected by the said compounds.
HBDH) should be ≤ 12, as predicted by the rule. TPSA is the topological
polar surface area and it is linked with poor membrane permeability and
low blood–brain barrier penetration. An ideal TPSA value is > 140 Å2.
The overall data given in the table shows that all the molecules meet the
criteria of Lipinski’s and Veber’s rules of good drug-likeness property
[36,37].
2.4. ADME studies
MedChem Designer software ver 3.0 was used for the determination
of pharmacological ADME (absorption, distribution, metabolism, and
excretion) properties of the compounds in search for ‘lead’ molecules as
proposed by Lipinski’s rule of five [29] and the calculated data is given
in Table 5. According to the rule, the lipophilicity of drug is determined
by logP, its value < 5.0 predicts a good partition coefficient. S + logP
and MlogP are octanol–water distribution coefficients; their values < 5
indicate that these molecules have drug-like properties. Lower numbers
of hydrogen bonds and higher logD, logP values predict higher
bioavailability of drugs. When there are > 5 hydrogen bond donors, the
sum of N and O atoms is > 10, log P is > 5 and molecular weight is >
500, the Lipinski’s rule is violated and poor absorption of the dug takes
place. Further, the sum of hydrogen bond donor and acceptor (M_NO +
2.5. Docking studies
The docking was performed from the stated softwares and the results
for the compounds 6a-o, were found to be positive in correspondence
with the standard quercetin and even better than the standard against
the all the used enzymes (5-LOX human, 15-LOX human; 15-LOX soy-
bean). The arachidonic acid as a substrate is a hydrophobic and flexible
molecule which easily fits in the catalytic site of the enzyme. The cat-
alytic center of 5-LOX mainly consists of the hydrophobic amino acids
Leu368, Ile406, Ala410, Leu414, Ile415, Leu691, Val604 and Leu607.
The iron atom is located at the middle of the active pocket. It is stabilized
Table 7
The docking score, inhibition constant (Ki) & nature of binding interaction for 6a-o with 15-LOX human.
Ligands/
Compds
Binding
affinity
Ki
Nature of binding interactions
(nMol)
–
–
6a
ꢀ 8.3
0.812
Leu201-Me(C H), Asp202-Me(C H), Arg429-Oxa.(
Leu610-Ar( -Alkyl).
Phe192-Oxa( ),S(
-Alkyl), Leu420-Ar(
Ala117-cyclohex.(Alkyl), Arg429-Oxa.(
Ala13-Pip.(Alkyl), Phe84-Pip.( -Alkyl),S(
Lys180-Ar( -Alkyl), Arg407-Oxa.( -anion),Oxa(H-B).
-anion), Ala144-Ar(
-S), Arg407-Ar( -Alkyl),Me(Alkyl).
-Alkyl), Glu141-Oxa.( -anion), Arg145-Oxa.(
π
-anion), Leu415-Ar(
π
-Alkyl), Ala416-Ar(
π
-Alkyl), Leu420-Ar(
π
-Alkyl),
π
–
6b
ꢀ 8.2
0.961
π
-
π
π
-S), Lys196-C
-Alkyl), Leu610-Ar(
-anion),Oxa.(H-B), Leu389-Pip.(Alkyl), Arg407-Ar(
-S), Trp109-Oxa( ), Arg145-Ar( -anion), Arg177-Ar(C H), Ala179-Ar(
–
O(H-B), Leu201-Oxa.(
π-Alkyl), Arg429-Oxa.(π-anion), Leu415-Ar(π-Alkyl), Ala416-Ar
(π
π
π
-Alkyl).
6c
6d
ꢀ 9.7
ꢀ 9.7
0.076
0.076
π
π-anion).
–
π
π
π
-
π
π
π
-Alkyl),
π
π
–
–
6e
ꢀ 9.3
0.150
Val117-C O(H-B), Glu140-Ar(
Leu389-Me(Alkyl), Phe399-S(
Val117-Ar(
π
π-Alkyl),Pip.(Alkyl), Arg145-Oxa.(H-B), Met148-Oxa(π-π), Ile174-Me(Alkyl),
π
π
6f
ꢀ 10.1
ꢀ 9.8
0.039
0.064
π
π
π
-anion), Phe399-Pip.(Alkyl), Arg407-Ar(
π
-anion).
–
–
6g
Val117-C
Ile403-Ar(
Pro223-(
-Alkyl), Thr663-Oxa.(H-B), Leu669-Pip.(Alkyl).
Gln108-(H-B), Arg145-Oxa.( -anion), Ile174-Ar(
-Alkyl).
Ala144-Ar(
Glu140-Ar(
-Alkyl).
Leu201-N(H-B), Phe184-Me(
-Alkyl), Me(Alkyl), Ala609-Ar(
O(H-B), Ala144-Ar(
π
-Alkyl),Pip.(Alkyl), Arg145-Oxa.(H-B), Met148-Oxa(
π
-
π
), Ile174-Me(Alkyl), Phe399-S(π-S),
π
-alkyl), Arg407-Ar(
π-Alkyl).
–
–
–
–
6h
6i
ꢀ 9.1
0.210
0.039
π
-Alkyl), Ala224-C O(H-B), His227-Me(Alkyl), Ala554-Ar(
π
-Alkyl), Ala558-Ar(
π-Alkyl), Glu613-C O(H-B), Pro661-Ar
(
π
ꢀ 10.1
π
π-Alkyl), His394-(H-B), Phe399-Pip(π-Alkyl), Ile403-Pip.(alkyl), Arg407-Ar
(π
6j
ꢀ 9.9
0.054
0.023
π
-Alkyl), Arg145-Oxa.(
π
-anion), Ile174-Ar(
π
-Alkyl), Ile403-Pip.(alkyl), Arg407-Ar(
π-Alkyl).
6k
ꢀ 10.4
π
-Alkyl), Ala144-Ar( -Alkyl),Me(Alkyl), Arg145-Oxa.(π
π
-anion), His394-Oxa.(H-B), Leu403-Pip.(Alkyl), Arg407-Ar
(π
6l
ꢀ 10.0
ꢀ 9.7
0.046
0.076
0.039
π
-Alkyl), Ala188-Me(Alkyl), Leu420-Me(Alkyl), Val426-Me(Alkyl), Arg429-Oxa.( -anion), Ala606-Ar
-Alkyl),Me(Alkyl), Leu610-Ar( -Alkyl).
-anion), Ala144-Pip.(alkyl), Arg145-Oxa.(π-anion), Ile403-Ar(π-Alkyl),
π
(π
π
–
π
6m
6n
Gln108-Oxa.(H-B), Gln137-Pip.(C H), Glu141-Oxa.(
Arg407-Ar( -alkyl).
Val117-Me(Alkyl), Ala144-Ar(
-Alkyl).
Gln108-Oxa.(H-B), Val117-Ar(
π
π
ꢀ 10.1
π-Alkyl),Me(Alkyl), Arg145-Oxa.(π-anion), Ile174-Ar(π-Alkyl), His394-Oxa.(H-B), Arg407-Ar
(π
6o
ꢀ 9.7
ꢀ 9.0
0.076
0.249
π
-Alkyl), Arg145-Oxa.(
π
-anion), His394-Oxa.(H-B), Leu389-Pip.(alkyl), Arg407-Ar(
π
-Alkyl).
Quercetin
Cys106-O(unfav), Tyr107-O(unfav), Arg145-O(H-B), Ile174-Ar(
π-alkyl), Phe399-Ar(π-π), Ile403-Ar(π-alkyl), Thr406-OH(unfav),
Tyr408-OH(unfav), Thr409-OH(H-B).
8

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
Fig. 5. 2D and 3D binding affinity of 6 k in 15-LOX human.
Table 8
The docking score, inhibition constant (Ki) & nature of binding interaction for 6a-o with 15-LOX soybean.
Ligands/
Compds
Binding
affinity
Ki
Nature of binding interactions
(nMol)
6a
6b
6c
ꢀ 7.1
ꢀ 7.2
ꢀ 8.7
6.202
5.238
0.416
Pro432-Pip.(Alkyl),Oxa.(
Pro432-Pip.(Alkyl),Oxa.(
π
π
-alkyl), Pro435-Ar(
π
π
-alkyl), Val589-Oxa.(
π
-alkyl), Asp592-Oxa.(
π
-anion).
-alkyl), Pro435-Ar(
-alkyl), Val588-Ar( -alkyl), Val589-Oxa.(
π
π
-alkyl), Asp592-Oxa.(
π
-anion).
–
–
–
Met19-Ar(
π
-alkyl), Leu89-Cyclohex.(Alkyl), Gln96-Oxa.-N-(C H), Val128-Oxa.(
π
-alkyl), Ser129-S(H-B),C O(H-B), Thr131-Me
–
(C H), Leu267-Ar(
Val26-Ar( -alkyl), Lys196-Ar(
Leu376-Me(Alkyl), Asp431-Pip.(C H), Pro432-Oxa.(
Ar( -anion), Arg607-Me(alkyl).
Arg378-Oxa.( -anion), Lys381-Me(Alkyl), Asp382-Ar(
Leu389-Pip.(Alkyl), Pro432-Oxa.(Alkyl), Val588-Pip.(Alkyl),Ar(
-anion).
–
– –
–
Met19-Ar(π-S), Thr91-N(H-B), Gln96-Oxa.(C H), Val128-Oxa.(π-alkyl), Ser129-C O(H-B),S(H-B), Thr131-Ar(C H), Leu267-Ar
π-alkyl).
6d
6e
ꢀ 8.6
ꢀ 8.3
0.492
0.817
π
π-alkyl), Leu258-Pip.(alkyl), Ala263-Pip.(alkyl), Phe272-Ar(π-π).
–
π
-alkyl), Pro435-Ar(π-alkyl), Val588-Ar(π-π), Val589-Oxa.(π-Alkyl), Lys606-
π
–
6f
ꢀ 8.5
ꢀ 8.4
0.583
0.690
π
π
-anion), Arg386-Ar(
π
-anion), Asp603-Pip.(C H),Ar(
π-anion).
6g
π
-
π
), Val589-Oxa.(π
-Alkyl), Trp593-Me(Alkyl), Lys606-Ar
(π
6h
6i
ꢀ 7.7
ꢀ 8.8
ꢀ 8.3
ꢀ 8.7
2.251
0.351
0.817
0.416
(
π
-alkyl), Arg592-N(unfav.).
–
–
π-π), Ser129-S(H-B), Thr131-Ar(C H), His142-
Met19-Ar(
Me(alkyl).
Met19-Ar(
π
-S), Leu24-Ar(
π
-alkyl), Thr91-N(H-B), Gln96-Oxa.(C H), Phe126-Ar.(
-alkyl), Leu89-Me(alkyl), Thr91-N(H-B), Thr131-Ar.(
–
-alkyl), Leu89-Me(alkyl), Thr91-N(H-B), Phe126-Ar.(π-π), Thr131-Ar.(C H), His142-Me(alkyl),
–
–
π-π), Arg552-Ar(π-anion), Asn788-C O(H-B),
6j
π
-S), Leu24-Ar(
π
Pro789-Pip.(Alkyl).
6k
Met19-Ar(
π
-S), Leu24-Ar(
π
–
–
Pro789-Pip.(Alkyl). Gln96-Oxa.(C H), Ser129-S(H-B), Thr131-Ar(C H), Lys388-Ar(alkyl), Leu389-Pip.(Alkyl), Asp431-Ar
–
–
(
π
-anion), Pro435-Me(alkyl), Val588-Ar(alkyl), Asp592-Ar(
π
-anion). Arg552-Ar(
π
-anion), Asn788-C O(H-B).
6l
ꢀ 8.3
0.817
Lys196-Ar(
π
-alkyl),Me(alkyl), Glu197-N(unfav.), Trp198-Me(alkyl), Ala263-Ar(
π
-alkyl),Pip.(Alkyl), Phe264-Ar(π-π), Asn556-Pip.
–
C
H).
–
π-π), Asn556-Pip.C H).
6 m
ꢀ 8.7
ꢀ 8.3
ꢀ 8.4
ꢀ 7.1
0.416
0.817
0.690
6.202
Lys196-Ar(
Lys196-Ar(
π
-alkyl),Me(alkyl), Glu197-N(unfav.), Ala263-Ar(
π
π
-alkyl), Pip.(Alkyl), Phe264-Ar(
-alkyl).
6n
π
-alkyl),Me(alkyl), Trp198-Me(alkyl), Ala263-Ar(
6o
Val26-Ar(
π
π
-alkyl), Arg195-N(H-B), Lys196-Ar(π-alkyl), Ala263-Pip.(Alkyl), Phe272-Ar(π-π).
–
–
Quercetin
Val26-Ar(
-alkyl),OH(unfav.), Glu197-OH(C H), Arg195-N(H-B), Tyr257-OH(C H), Leu258-OH(H-B),Ar(
π-alkyl), Ala263-Fur.
(Alkyl), Ar(
π
-alkyl), Asn556-OH(H-B).
by coordination bonds with His367, His372, His550 and Asn554. The
Phe177 also participates in complex stabilization and interacts with the
carboxylic group of arachidonic acid [38]. The catalytic sites of 15-LOX
are almost same as far as amino acids are concerned. The binding affinity
and the inhibitory concentration for the maximum compounds were
found enhanced than that of the standard. The compound 6n has the
best binding affinity and the inhibitory constant against 5-LOX human, i.
e., ꢀ 9.1 and 0.212 while the standard quercetin has ꢀ 8.0 and 1.356
(Table 6; Fig. 4) whereas, the binding affinity and the inhibitory con-
stant for compound 6 k against 15-LOX human was found to be the best,
Fig. 6. 2D and 3D binding affinity of 6i in 15-LOX soybean.
9

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
Fig. 7. 2D and 3D binding affinity of standard quercetin in 15-LOX soybean.
i.e., ꢀ 10.4 and 0.023 while the standard quercetin had ꢀ 9.0 and 0.249
(Table 7; Fig. 5). The binding affinity and the inhibitory constant against
15-LOX soybean for 6i was found to be the best, i.e., ꢀ 8.8 and 0.351
while compared with standard quercetin it is ꢀ 7.1 and 6.202 (Table 8;
Figs. 6-7). From these results it has been found that the oxadiazole entity
revealed better bonding with Arg and Phe residues while Leu and Ile,
Ala, Val, were more prone to other parts of the synthetic compounds.
Similarly, in the standard quercetin, the residues bound were Val, Arg,
Phe and Ile. However, Phe and Tyr at other positions are also active in
both the sequences that lead to different interactions against a same
inhibitor. During docking, it was therefore observed that the binding
with the tested compounds as well as the standard quercetin, the strong
hydrogen bonding was mostly observed due to the active hydrogen as
well as oxygen from the oxadiazole group. The bonding was most likely
positions as well as nature of substituents linked to phenyl ring effect 15-
LOX inhibition which was further supported by molecular docking
studies with all three LOX types. The present work enhances the credi-
bility of the oxadiazole ring system in search for new drugs as anti-LOX
agents. Further work is continued on the design, synthesis and deriva-
tization of active compounds of oxadiazole ring systems in search for
more ‘leads’ as LOX inhibitors.
4. Experimental
4.1. Materials
All the chemical reagents and solvents of analytical grade were
copped from local vendor of Sigma, Aldrich and Alfa Aesar. The 1H- and
13C NMR spectra were logged on Bruker instrument operating at 400 and
100 MHz, respectively, using tetramethylsilane as internal standard. The
IR spectra were retrieved on Shimadzu 460 FTIR spectrometer using KBr
disk. The mass spectra were regulated on JMSA 500 mass spectrometer
and JMS H × 110 spectrometer with data system. Melting points were
opted by using GallenKemp electro-thermal apparatus.
due to the benzene ring including
o while the interactions were dominantly
they were -cation, -anion and sometimes hydrogen bonding in-
π
-
π
and
π
-alkyl type present in the 6a-
π
-type but more specifically
π
π
teractions. The bonding including π-π, π-alkyl and hydrogen bond in-
dicates the presence of better results [39].
3. Conclusion
4.2. Synthesis of ethyl 1-phenylcarbamoylpiperidine-4-carboxylate (1)
It has been observed that 1,3,4-oxadiazoles having toxophoric
–
– – –
correlation form hydrogen bonding with various receptors
N
C
O
–
Ethyl piperidine-4-carboxylate (a; 0.03 mol, 4.6 mL) was taken on a
watch glass and phenyl isocyanate (0.03 mol, 3.2 mL) was added
dropwise with constant rubbing and it was continued for 15–20 min
until the formation of white solid. The physical and spectroscopic data
compound 1 is as follows:
molecules that result in increased biological activities, the property
exploited in the process of drug discovery. A plethora of literature is
available and being published on the drug-ability of these classes of
compounds. Our group has been interested in the development of
pharmacophores against several inflammatory diseases including can-
cer. In conclusion, a series of 2-(5-(1-phenylcarbamoylpiperidine)-4H-
1,3,4-oxadiazol-3-ylthio)acetamide scaffolds (6a-o) were designed,
synthesized and characterized and evaluated for in vitro inhibition
studies against soybean 15-LOX. Compounds exhibited potent to mod-
erate and poor inhibitory potential in the decreasing order of inhibition
as: 6g > 6b > 6a > 6l > 6k > 6 m > 6j > 6o > 6h > 6f > 6i > 6n > 6c >
6e > 6d. Compounds 6g, 6b, 6a and 6 l were potent 15-LOX inhibitors
with less toxicity and potential drug-likeness properties. This was
evident in the relative expression of 5-LOX gene found in human MNCs
after treatment with only three selected molecules which down-
regulated the enzyme activity. These results demonstrate that the said
‘lead’ compounds active in in vitro assay against soybean LOX were also
active against human 5-LOX expressed in human MNCs. Flow cytometry
analysis of selected compounds 6a, 6j and 6n revealed 79.5–88.51%
early apoptotic changes in MNCs at their respective IC50 values
compared with the control quercetin. SAR studies show that the
White solid; Yield: 99%; M.P.: 105–108 ◦C; IR (KBr disk, cmꢀ 1): 3394
–
–
–
–
(N H), 3040 (Ar-H), 2952(C H), 1733, 1661 (C O), 1615–1562 (Ar-
–
1
–
– –
C
C), 1261 (C O, C N); H NMR (400 MHz, CDCl3, ppm): δ 1.28 (3H,
t, J = 7.0 Hz, CH3-CH2-O), 1.87 (4H, m, H-3,5), 3.31 (1H, m, H-4), 3.37
(2H, m, H-2α,6α), 4.12 (2H, q, J = 7.0 Hz, CH3-CH2-O), 4.31 (2H, m, H-
2β,6β), 7.18 (1H, t, J = 7.5 Hz, H-4ʹ), 7.41 (2H, t, J = 7.5 Hz, H-3ʹ,5ʹ),
7.60 (2H, d, J = 7.5 Hz, H-2ʹ,6ʹ); 13C NMR (100 MHz, CD3OD): δ 14.2
(CH3), 28.3 (C-3,5), 32.2 (C-4), 46.5 (C-2,6), 62.2 (CH2), 121.4 (C-2ʹ,6ʹ),
–
128.1 (C-4ʹ), 128.7 (C-3ʹ,5ʹ), 139.5 (C-1ʹ), 158.2 (C O), 175.6 (COOEt);
–
HR-EI-MS (m/z): 276.1491 [M]+ calculated for C15H20N2O3; 276.1473.
4.3. Synthesis of 1-phenylcarbamoylpiperidine-4-carboxamide (2)
Ethyl 1-phenylcarbamoylpiperidine-4-carboxylate (1; 0.028 mol,
7.9 g) was taken in 100 mL round bottom flask and 50 mL of hydrazine
hydrate (80%) was added slowly with constant stirring at room tem-
perature which further continued for 3–4 hrs. The white precipitates
10

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
were formed which were filtered, washed with cold water and dried. The
3ʹ,5ʹ), 34.5 (C-4ʹ), 36.9 (C-2ʹʹʹ), 42.2 (C-1ʹʹʹʹ), 43.9 (C-1′′′′′), 44.5 (C-2ʹ,6ʹ),
122.2 (C-2ʹʹ,6ʹʹ), 124.1 (C-4ʹʹ), 129.6 (C-3ʹʹ,5ʹʹ), 140.9 (C-1ʹʹ), 157.8
physical and spectroscopic data of 2 is as follows:
◦
–
White shiny powder; Yield: 98%; M.P.: 160–168 C; IR (KBr disk,
(C O), 165.8 (C-2), 167.6 (C-5), 171.4 (C-1ʹʹʹ); HR-EI-MS (m/z):
–
cmꢀ 1): 3395, 3363 (N H), 3041 (Ar-H), 2953 (C H), 1681, 1660
417.1854 [M]+ calculated for C20H27N5O3S; 417.1834.
–
–
1
–
–
–
–
–
(C O), 1615–1561 (Ar-C C), 1243 (C N); H NMR (400 MHz, CDCl3,
ppm): δ 1.88 (4H, m, H-3,5), 3.32 (1H, m, H-4), 3.38 (2H, m, H-2
α
,6α
),
4.6.2. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-n-propylacetamide (6b)
4.30 (2H, m, H-2β,6β), 7.18 (1H, t, J = 7.5 Hz, H-4ʹ), 7.42 (2H, t, J = 7.5
Hz, H-3ʹ,5ʹ), 7.60 (2H, d, J = 7.5 Hz, H-2ʹ,6ʹ); 13C NMR (100 MHz,
White amorphous powder; Yield: 96%; M.P.:149–150˚C; IR (KBr)
–
– –
νmax: 3363 (N H), 3033 (Ar-H), 2932 (C H), 1683, 1660 (C O),
–
CD3OD): δ 29.5 (C-3,5), 33.2 (C-4), 47.2 (C-2,6), 121.5 (C-2ʹ,6ʹ), 128.1
1
–
–
– –
–
(C-4ʹ), 128.7 (C-3ʹ,5ʹ), 139.2 (C-1ʹ), 158.2 (C O), 178.1 (CONH); HR-EI-
1619–1553 (Ar-C C, C N), 1257 (C N); H NMR (400 MHz, CD3OD):
δ 0.90 (3H, t, J =–7.4 H–z, H-3′′′′
MS (m/z): 262.1449 [M]+ calculated for C15H18N4O2; 262.1429.
), 1.52 (2H, hexet, J = 7.4 Hz, H-2′′′′),
1.82 (2H, m, H-3αʹ,5αʹ), 2.11 (2H, m, H-3βʹ,5βʹ), 3.10 (2H, m, H-2αʹ,6αʹ),
3.16 (2H, t, J = 7.4 Hz, H-1ʹʹʹʹ), 3.19 (1H, m, H-4ʹ), 3.93 (2H, s, H-2′′′),
4.15 (2H, m, H-2βʹ,6βʹ), 7.00 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.24 (2H, t, J =
7.5 Hz, H-3ʹʹ,5ʹʹ), 7.33 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ); 13C NMR (100 MHz,
CD3OD): δ 11.6 (C-3ʹʹʹʹ), 23.1 (C-2ʹʹʹʹ), 29.6 (C-3ʹ,5ʹ), 34.1 (C-4ʹ), 36.1 (C-
2ʹʹʹ), 42.5 (C-1ʹʹʹʹ), 44.1 (C-2ʹ,6ʹ), 121.8 (C-2ʹʹ,6ʹʹ), 123.8 (C-4ʹʹ), 129.3 (C-
4.4. Synthesis of 5-(1-phenylcarbamoylpiperidine)-4H-1,3,4-oxadiazole-
2-thiol (3)
The 1-phenylcarbamoylpiperidine-4-carboxamide (2; 0.027 mol, 7.3
g) was dissolved in 50 mL absolute ethanol in 100 mL round bottom
flask. 2.66 mL (0.054 mol) of carbon disulfide was added to the flask
followed by the addition of 1.5 g (0.027 mol) of potassium hydroxide.
The mixture was refluxed for 4–5 hrs with stirring. The contents of re-
action was poured in ice cold water pH 5–6 was maintained by using
dilute HCl. The white precipitates were appeared which were filtered,
washed and dried. The physical and spectroscopic data of 3 is as follows:
–
–
3ʹʹ,5ʹʹ), 140.3 (C-1ʹʹ), 157.3 (C O), 165.0 (C-2), 168.3 (C-5), 171.0 (C-
1ʹʹʹ); HR-EI-MS (m/z): 403. 1699 [M]+ calculated for C19H25N5O3S; 403.
1678.
4.6.3. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-cyclohexylacetamide (6c)
White powder; Yield: 97%; M.P.:182–185 C; IR (KBr disk, cmꢀ 1):
◦
White amorphous powder; Yield: 98%; M.P.:162–165˚C; IR (KBr)
–
–
–
–
–
–
νmax: 3364 (N H), 3030 (Ar-H), 2933 (C H), 1682, 1658 (C O),
3365 (N
H), 1661 (C O), 1617–1551 (Ar-
–
– –H), 3031 (Ar-H), 2927 (C
–
1
1
– –
–
–
1611–1557 (Ar-C C, C N), 1262 (C N); H NMR (400 MHz, CD3OD):
– –
C
C, C N), 1233 (C N); H NMR (400 MHz, CDCl3, ppm): δ 1.82 (4H,
m–, H-3ʹ,–5ʹ), 3.34 (1H, m, H-4ʹ), 3.38 (2H, m, H-2
δ 1.19 (2H, m, H-4ʹʹʹʹ), 1.31 (4H, m, H-3ʹʹʹʹ,5ʹʹʹʹ), 1.60 (4H, m, H-2ʹʹʹʹ,6ʹʹʹʹ),
α
ʹ,6αʹ), 4.13 (2H, m H-
1.76 (1H, m, H-1ʹʹʹʹ), 1.86 (2H, m, H-3αʹ,5αʹ), 2.12 (2H, m, H-3βʹ,5βʹ),
2βʹ,6βʹ), 7.00 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.29 (2H, t, J = 7.5 Hz, H-3ʹʹ,5ʹʹ),
7.35 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ); 13C NMR (100 MHz, CD3OD): δ 29.0
3.12 (2H, m, H-2αʹ,6α
ʹ), 3.20 (1H, m, H-4ʹ), 3.92 (2H, s, H-2′′′), 4.16 (2H,
m, H-2βʹ,6βʹ), 7.00 (1H, t, J = 7.3 Hz, H-4ʹʹ), 7.24 (2H, t, J = 7.3 Hz, H-
3ʹʹ,5ʹʹ), 7.34 (2H, d, J = 7.3 Hz, H-2ʹʹ,6ʹʹ); 13C NMR (100 MHz, CD3OD): δ
25.7 (C-2ʹʹʹʹ,6ʹʹʹʹ), 26.3 (C-4ʹʹʹʹ), 29.2 (C-1ʹʹʹʹ), 29.7 (C-3ʹʹʹʹ,5ʹʹʹʹ), 33.3 (C-
3ʹ,5ʹ), 34.2 (C-4ʹ), 36.4 (C-2ʹʹʹ), 44.2 (C-2ʹ,6ʹ), 122.0 (C-2ʹʹ,6ʹʹ), 123.9 (C-
(C-3ʹ,5ʹ), 31.4 (C-4ʹ), 44.1 (C-2ʹ,6ʹ), 121.4 (C-2ʹʹ,6ʹʹ), 124.0 (C-4ʹʹ), 126.9
–
–
(C-3ʹʹ,5ʹʹ), 140.4 (C-1ʹʹ), 157.7(C O), 158.5 (C-5), 162.3 (C-2); HR-EI-
MS (m/z): 304.0994 [M]+ calculated for C14H16N4O2S; 304.0970.
4.5. General procedure for the synthesis of compounds (5a-o)
–
–
4ʹʹ), 129.3 (C-3ʹʹ,5ʹʹ), 140.4 (C-1ʹʹ), 157.5 (C O), 165.2 (C-2), 167.6 (C-
5), 171.1 (C-1ʹʹʹ); HR-EI-MS (m/z): 443.2012 [M]+ calculated for
C
22H29N5O3S; 443.1991.
Computed amounts of alkyl/aralkyl/aryl amines (4a-o, 0.01 mol)
were added separately in a quick fit Erlenmeyer flask already having
20% aqueous Na2CO3 solution to set the pH 9–10. The bromoacetyl
bromide (0.01 mol) was added slowly and dropwise into the flask over
2–5 min with continuous vigorous shaking till the formation of pre-
cipitates followed by further stirring in order to get homogenous pre-
cipitates for 1–2 hrs. The solid product was filtered, washed with cold
water and dried to get the respective electrophile(s), N-alky/aralkyl/
aryl substituted-2-bromoacetamides (5a-o).
4.6.4. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-benzylacetamide (6d)
White amorphous powder; Yield: 98%; M.P.: 178–180˚C; IR (KBr)
–
–
–
νmax: 3349 (N H), 3033 (Ar-H), 2935 (C H), 1680, 1662 (C O),
–
1
– –
–
1617–1550 (Ar-C C, C N), 1261 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.81 (2H, m, H-3
αʹ,5αʹ), 2.08 (2H, m, H-3βʹ,5βʹ), 3.07 (2H, m, H-
2α
ʹ,6αʹ), 3.16 (1H, m, H-4ʹ), 3.98 (2H, s, H-7ʹʹʹʹ), 4.14 (2H, m, H-2βʹ,6βʹ),
4.39 (2H, s, H-2′′′), 7.00 (1H, t, J = 7.3 Hz, H-4ʹʹ), 7.23 (2H, t, J = 7.3 Hz,
H-3ʹʹ,5ʹʹ), 7.26 (3H, m, H-3ʹʹʹʹ-5ʹʹʹʹ), 7.31 (2H, m, H-2ʹʹʹʹ,6ʹʹʹʹ), 7.34 (2H, d, J
= 7.3 Hz, H-2ʹʹ,6ʹʹ); 13C NMR (100 MHz, CD3OD): δ 29.5 (C-3ʹ,5ʹ), 34.1
(C-4ʹ), 36.1 (C-2ʹʹʹ), 44.1 (C-2ʹ,6ʹ), 44.4 (C-7ʹʹʹʹ), 121.8 (C-2ʹʹ,6ʹʹ), 123.8 (C-
4ʹʹ), 128.0 (C-4ʹʹʹʹ), 128.3 (C-3ʹʹ,5ʹʹ), 129.2 (C-2ʹʹʹʹ,3ʹʹʹʹ,5ʹʹʹʹ,6ʹʹʹʹ), 138.7 (C-
4.6. General method for the synthesis of compounds (6a-o)
In a 50 mL round bottom flask, 0.2 g (0.6 mmol) of compound 3 was
dissolved in absolute ethanol and KOH (0.6 mmol, 0.036 g) was added,
the mixture was stirred for 30 min at room temperature. The electro-
philes (5a-o) were added slowly and separately to the mixture and
further stirrer for 4–5 hrs. Excess amount of cold distilled water was
added and the precipitates of the final products, (6a-o) were formed,
separately, which were filtered, washed and dried.
–
–
1ʹʹʹʹ), 140.2 (C-1ʹʹ), 157.3 (C O), 164.9 (C-2), 168.3 (C-5), 171.0 (C-1ʹʹʹ);
HR-EI-MS (m/z): 451.1698 [M]+ calculated for C23H25N5O3S; 451.1678.
4.6.5. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-(2-methylphenyl)acetamide (6e)
White amorphous powder; Yield: 93%; M.P.: 150–152˚C; IR (KBr)
–
–
–
νmax: 3353 (N H), 3032 (Ar-H), 2933 (C H), 1682, 1663 (C O),
–
4.6.1. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
1
– –
–
1617–1550 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
– –
N,N-diethylacetamide (6a)
δ 1.82 (2H, m, H-3
αʹ,5αʹ), 2.10 (2H, m, H-3βʹ,5βʹ), 2.23 (3H, s, CH3), 3.09
White amorphous powder; Yield: 99%; M.P.: 81–82˚C; IR (KBr) νmax
:
–
–
–
–
(2H, m, H-2 ʹ,6αʹ), 3.17 (1H, m, H-4ʹ), 4.12 (2H, m, H-2βʹ,6βʹ), 4.14 (2H,
α
3362 (N H), 3030 (Ar-H), 2933 (C H), 1680, 1659 (C O),
1
s, H-2′′′), 6.99 (1H, t, J = 7.3 Hz, H-4ʹʹ), 7.09 (1H, t, J = 7.4 Hz, H-4ʹʹʹʹ),
7.15 (1H, t, J = 7.4 Hz, H-6ʹʹʹʹ), 7.17 (1H, t, J = 7.4 Hz, H-5ʹʹʹʹ), 7.24 (2H,
t, J = 7.3 Hz, H-3ʹʹ,5ʹʹ), 7.31 (2H, d, J = 7.3 Hz, H-2ʹʹ,6ʹʹ), 7.44 (1H, d, J =
7.4 Hz H-3ʹʹʹʹ); 13C NMR (100 MHz, CD3OD): δ 18.0 (CH3), 29.5 (C-3ʹ,5ʹ),
34.0 (C-4ʹ), 36.5 (C-2ʹʹʹ), 44.0 (C-2ʹ,6ʹ), 121.7 (C-2ʹʹ,6ʹʹ), 123.7 (C-4ʹʹ),
125.5 (C-6ʹʹʹʹ), 126.9 (C-4ʹʹʹʹ), 127.0 (C-5ʹʹʹʹ), 129.2 (C-3ʹʹ,5ʹʹ), 131.2 (C-
– –
–
1615–1550 (Ar-C C, C N), 1250 (C N); H NMR (400 MHz, CD3OD):
δ 1.13 (3H, t, J =–7.2 H–z, H-2′′′′
), 1.27 (3H, t, J = 7.2 Hz, H-2′′′′′), 1.81
(2H, m, H-3αʹ,5αʹ), 2.12 (2H, m, H-3βʹ,5βʹ), 3.14 (2H, m, H-2αʹ,6αʹ), 3.24
(1H, m, H-4ʹ), 3.41 (2H, q, J = 7.2 Hz, H-1ʹʹʹʹ), 3.49 (2H, q, J = 7.2 Hz, H-
1′′′′′), 4.16 (2H, m, H-2βʹ,6βʹ), 4.36 (2H, s, H-2′′′), 7.01 (1H, t, J = 7.7 Hz,
H-4ʹʹ), 7.26 (2H, t, J = 7.7 Hz, H-3ʹʹ,5ʹʹ), 7.35 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ);
13C NMR (100 MHz, CD3OD): δ 13.1 (C-2′′′′), 14.3 (C-2′′′′′), 30.1 (C-
–
–
3ʹʹʹʹ), 132.7 (C-2ʹʹʹʹ), 135.8 (C-1ʹʹʹʹ), 140.1 (C-1ʹʹ), 157.1 (C O), 165.0 (C-
11

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
2), 167.1 (C-5), 170.9 (C-1ʹʹʹ); HR-EI-MS (m/z): 451.1699 [M]+ calcu-
3ʹ,5ʹ), 35.5 (C-4ʹ), 36.1 (C-2ʹʹʹ), 43.0 (C-2ʹ,6ʹ), 119.7 (C-2ʹʹ,6ʹʹ), 123.3 (C-
4ʹʹ), 125.2 (C-4ʹʹʹʹ), 127.2 (C-6ʹʹʹʹ), 128.2 (C-3ʹʹ-5ʹʹ), 131.1 (C-2ʹʹʹʹ), 135.6
lated for C23H25N5O3S; 451.1678.
–
(C-3ʹʹʹʹ), 137.1 (C-5ʹʹʹʹ), 140.5 (C-1ʹʹʹʹ), 140.6 (C-1ʹʹ), 154.9 (C O), 162.8
–
4.6.6. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-3-methylphenylacetamide (6f)
(C-2), 165.1 (C-5), 169.7 (C-1′′′); HR-EI-MS (m/z): 465.1854 [M]+
calculated for C24H27N5O3S; 465.1834.
White amorphous powder; Yield: 94%; M.P.: 158–160˚C; IR (KBr)
–
–
–
νmax: 3350 (N H), 3032 (Ar-H), 2935 (C H), 1680, 1660 (C O),
4.6.10. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-
ylthio)-N-2,4-dimethylphenylacetamide (6j)
–
1
– –
–
1618–1558 (Ar-C C, C N), 1263 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.82 (4H, m, H-3
α
ʹ,5α
ʹ), 2.10 (4H, m, H-3βʹ,5βʹ), 2.30 (3H, s, CH3), 3.10
White amorphous powder; Yield: 95%; M.P.: 166–168˚C; IR (KBr)
–
– –
νmax: 3351 (N H), 3032 (Ar-H), 2935 (C H), 1680, 1660 (C O),
–
(2H, m, H-2 ʹ,6
α
α
ʹ), 3.19 (1H, m, H-4ʹ), 4.11 (2H, m, H-2βʹ,6βʹ), 4.14 (2H,
1
s, H-2′′′), 6.90 (1H, d, J = 8.0 Hz, H-4ʹʹʹʹ), 7.00 (1H, t, J = 8.0 Hz, H-4ʹʹ),
7.17 (1H, t, J = 8.0 Hz, H-5ʹʹʹʹ), 7.24 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.32
(1H, d, J = 8.0 Hz, H-4ʹʹʹʹ), 7.34 (2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), 7.37 (1H, s,
H-2ʹʹʹʹ); 13C NMR (100 MHz, CD3OD): δ 21.6 (CH3), 29.8 (C-3ʹ,5ʹ), 34.3
(C-4ʹ), 37.3 (C-2ʹʹʹ), 44.2 (C-2ʹ,6ʹ), 118.1 (C-2ʹʹʹʹ), 121.6 (C-4ʹʹʹʹ), 122.0 (C-
2ʹʹ,6ʹʹ), 124.0 (C-4ʹʹ), 126.2 (C-6ʹʹʹʹ), 129.4 (C-3ʹʹ,5ʹʹ), 129.6 (C-5ʹʹʹʹ), 139.0
1617–1559 (Ar-C C, C N), 1263 (C N); H NMR (400 MHz, CD3OD):
– –
– –
–
δ 1.83 (2H, m, H-3αʹ,5αʹ), 2.11 (2H, m, H-3βʹ,5βʹ), 2.19 (3H, s, CH3), 2.26
(3H, s, CH3), 3.11 (4H, m, H-2αʹ,6αʹ), 3.20 (1H, m, H-4ʹ), 4.13 (4H, m, H-
2βʹ,6βʹ), 4.16 (2H, s, H-2′′′), 6.95 (1H, s, H-3ʹʹʹʹ), 6.97 (1H, d, J = 8.0 Hz,
H-6ʹʹʹʹ), 6.98 (1H, d, J = 8.0 Hz, H-4ʹʹʹʹ), 7.00 (1H, t, J = 7.7 Hz, H-4ʹʹ),
7.24 (2H, t, J = 7.7 Hz, H-3ʹʹ,5ʹʹ), 7.34 (2H, d, J = 7.7 Hz, H-2ʹʹ,6ʹʹ); 13
C
–
–
(C-3ʹʹʹʹ), 139.7 (C-1ʹʹʹʹ), 140.6 (C-1ʹʹ), 157.5 (C O), 165.1 (C-2), 166.8 (C-
NMR (100 MHz, CD3OD): δ 18.0 (CH3), 21.0 (CH3), 29.8 (C-3ʹ,5ʹ), 34.3
(C-4ʹ), 36.6 (C-2ʹʹʹ), 44.2 (C-2ʹ,6ʹ), 122.0 (C-2ʹʹ,6ʹʹ), 123.9 (C-4ʹʹ), 126.1
(C-6ʹʹʹʹ), 127.7 (C-3ʹʹʹʹ), 129.4 (C-3ʹʹ,5ʹʹ), 132.0 (C-5ʹʹʹʹ), 133.4 (C-4ʹʹʹʹ),
5), 171.3 (C-1ʹʹʹ); HR-EI-MS (m/z): 451.1699 [M]+ calculated for
C
23H25N5O3S; 451.1678.
–
–
133.5 (2ʹʹʹʹ), 137.1 (C-1ʹʹʹʹ), 140.5 (C-1ʹʹ), 157.5 (C O), 165.1 (C-2),
4.6.7. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
167.6 (C-5), 171.3 (C-1′′′); HR-EI-MS (m/z): 465.1856 [M]+ calculated
N-2-ethylphenylacetamide (6g)
for C24H27N5O3S; 465.1834.
White amorphous powder; Yield: 96%; M.P.: 90–92˚C; IR (KBr) νmax
:
–
–
–
–
3350 (N H), 3032 (Ar-H), 2930 (C H), 1681, 1661 (C O),
4.6.11. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-
ylthio)-N-2,5-dimethylphenylacetamide (6k)
1
– –
–
1617–1550 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.15 (3H, t, J = 7.5 Hz, CH3-CH2), 1.83 (2H, m, H-3
αʹ,5α
ʹ), 2.11 (2H, m,
ʹ,6 ʹ),
White amorphous powder; Yield: 94%; M.P.: 172–175˚C; IR (KBr)
–
– –
νmax: 3352 (N H), 3031 (Ar-H), 2933 (C H), 1680, 1667 (C O),
–
H-3βʹ,5βʹ), 2.60 (2H, q, J = 7.5 Hz, CH3-CH2), 3.10 (2H, m, H-2
α
α
1
3.20 (1H, m, H-4ʹ), 4.13 (2H, m, H-2βʹ,6βʹ), 4.16 (2H, s, H-2′′′), 7.00 (1H,
t, J = 7.5 Hz, H-4ʹʹ), 7.14 (1H, d, J = 7.5 Hz, H-6ʹʹʹʹ), 7.18 (1H, t, J = 7.5
Hz, H-4ʹʹʹʹ), 7.21 (1H, d, J = 7.5 Hz, H-3ʹʹʹʹ), 7.24 (2H, t, J = 7.5 Hz, H-
3ʹʹ,5ʹʹ), 7.33 (2H, t, J = 7.5 Hz, H-2ʹʹ,6ʹʹ), 7.38 (1H, t, J = 7.5 Hz, H-5ʹʹʹʹ);
13C NMR (100 MHz, CD3OD): δ 14.8 (CH3), 25.0 (CH2), 29.7 (C-3ʹ,5ʹ),
34.2 (C-4ʹ), 36.6 (C-2ʹʹʹ), 44.1 (C-2ʹ,6ʹ), 121.9 (C-2ʹʹ,6ʹʹ), 123.9 (C-4ʹʹ),
126.7 (C-6ʹʹʹʹ), 127.1 (C-4ʹʹʹʹ), 127.7 (C-3ʹʹʹʹ), 129.3 (C-3ʹʹ,5ʹʹ), 129.6 (C-
1615–1550 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
– –
– –
–
δ 1.83 (2H, m, H-3
αʹ,5αʹ), 2.12 (2H, m, H-3βʹ,5βʹ), 2.19 (3H, s, CH3), 2.27
(3H, s, CH3), 3.11 (2H, m, H-2
α
ʹ,6αʹ), 3.21 (1H, m, H-4ʹ), 4.14(2H, m, H-
2βʹ,6βʹ), 4.17 (2H, s, H-2′′′), 6.93 (1H, d, J = 7.7 Hz, H-4ʹʹʹʹ), 6.99 (1H, t, J
= 7.3 Hz, H-4ʹʹ), 7.07 (1H, d, J = 7.7 Hz, H-3ʹʹʹʹ), 7.22 (1H, s, H-6ʹʹʹʹ), 7.24
(2H, t, J = 7.3 Hz, H-3ʹʹ,5ʹʹ), 7.35 (2H, d, J = 7.3 Hz, H-2ʹʹ,6ʹʹ); 13C NMR
(100 MHz, CD3OD): δ 17.6 (CH3), 21.0 (CH3), 29.8 (C-3ʹ,5ʹ), 33.3 (C-4ʹ),
36.7 (C-2ʹʹʹ), 44.2 (C-2ʹ,6ʹ), 120.0 (C-2ʹʹ,6ʹʹ), 124.0 (C-4ʹʹ), 126.6 (C-6ʹʹʹʹ),
128.0 (C-4ʹʹʹʹ), 129.4 (C-3ʹʹ,5ʹʹ), 130.2 (C-2ʹʹʹʹ), 131.3 (C-3ʹʹʹʹ), 136.0 (C-
–
–
5ʹʹʹʹ), 135.2 (C-2ʹʹʹʹ), 139.4 (C-1ʹʹʹʹ), 140.3 (C-1ʹʹ), 157.3 (C O), 165.1 (C-
2), 167.8 (C-5), 171.2 (C-1ʹʹʹ); HR-EI-MS (m/z): 465.1855 [M]+ calcu-
–
–
lated for C24H27N5O3S; 465.1834.
5ʹʹʹʹ), 136.9 (C-1ʹʹʹʹ), 140.5 (C-1ʹʹ), 157.5 (C O), 165.2 (C-2), 167.6 (C-5),
171.3 (C-1′′′); HR-EI-MS (m/z): 465.1855 [M]+ calculated for
4.6.8. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-4-ethylphenylacetamide (6h)
C24H27N5O3S; 465.1834.
White amorphous powder; Yield: 97%; M.P.: 165–170˚C; IR (KBr)
4.6.12. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-
ylthio)-N-2,6-dimethylphenylacetamide (6 l)
–
–
–
νmax: 3351 (N H), 3030 (Ar-H), 2937 (C H), 1680, 1668 (C O),
–
1
– –
–
1611–1557 (Ar-C C, C N), 1267 (C N); H NMR (400 MHz, CD3OD):
– –
White amorphous powder; Yield: 92%; M.P.: 176–179˚C; IR (KBr)
–
– –
νmax: 3351 (N H), 3033 (Ar-H), 2947 (C H), 1683, 1660 (C O),
–
δ 1.18 (3H, t, J = 7.6 Hz, CH3-CH2), 1.81 (2H, m, H-3
αʹ,5α
ʹ), 2.09 (2H, m,
ʹ,6 ʹ),
1
– –
–
1615–1554 (Ar-C C, C N), 1268 (C N); H NMR (400 MHz, CD3OD):
– –
H-3βʹ,5βʹ), 2.56 (2H, q, J = 7.6 Hz, CH3-CH2), 3.08 (2H, m, H-2
α
α
3.16 (1H, m, H-4ʹ), 4.10 (2H, s, H-2′′′), 4.13 (2H, m, H-2βʹ,6βʹ), 6.99 (1H,
t, J = 7.3 Hz, H-4ʹʹ), 7.12 (2H, d, J = 8.5 Hz, H-2ʹʹʹʹ,6ʹʹʹʹ), 7.23 (2H, t, J =
7.3 Hz, H-3ʹʹ,5ʹʹ), 7.31 (2H, d, J = 7.3 Hz, H-2ʹʹ,6ʹʹ), 7.43 (2H, d, J = 8.5
Hz, H-3ʹʹʹʹ,5ʹʹʹʹ); 13C NMR (100 MHz, CD3OD): δ 16.0 (CH3), 28.9 (CH2),
29.5 (C-3ʹ,5ʹ), 34.0 (C-4ʹ), 37.1 (C-2ʹʹʹ), 44.0 (C-2ʹ,6ʹ), 120.9 (C-2ʹʹʹʹ,6ʹʹʹʹ),
121.7 (C-2ʹʹ,6ʹʹ), 123.8 (C-4ʹʹ), 128.8 (C-3ʹʹʹʹ,5ʹʹʹʹ), 129.2 (C-3ʹʹ,5ʹʹ), 136.3
δ 1.82 (2H, m, H-3
αʹ,5αʹ), 2.12 (2H, m, H-3βʹ,5βʹ), 2.19 (6H, s, CH3), 3.13
(2H, m, H-2 ʹ,6αʹ), 3.24 (1H, m, H-4ʹ), 4.15 (2H, m, H-2βʹ,6βʹ), 4.22 (2H,
α
s, H-2′′′), 6.99 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.06(3H, s, H-3ʹʹʹʹ-5ʹʹʹʹ), 7.25 (2H,
t, J = 7.5 Hz, H-3ʹʹ,5ʹʹ), 7.34 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ); 13C NMR (100
MHz, CD3OD): δ 18.4 (CH3), 30.0 (C-3ʹ,5ʹ), 34.4 (C-4ʹ), 36.3 (C-2ʹʹʹ), 44.4
(C-2ʹ,6ʹ), 122.2 (C-2ʹʹ,6ʹʹ), 124.1 (C-4ʹʹ), 128.5 (C-4ʹʹʹʹ), 129.0 (C-3ʹʹʹʹ,5ʹʹʹʹ),
129.5 (C-3ʹʹ,5ʹʹ), 134.9 (C-1ʹʹʹʹ), 136.7 (2ʹʹʹʹ,6ʹʹʹʹ), 140.8 (C-1ʹʹ), 157.7
–
–
(C-4ʹʹʹʹ), 140.1 (C-1ʹʹʹʹ), 141.5 (C-1ʹʹ), 157.2 (C O), 165.0 (C-2), 166.3 (C-
5), 170.9 (C-1ʹʹʹ); HR-EI-MS (m/z): 465.1854 [M]+ calculated for
(C O), 165.2 (C-2), 167.8 (C-5), 171.6 (C1 ); HR-EI-MS (m/z):
′′′
–
–
C
24H27N5O3S; 465.1834.
465.1856 [M]+ calculated for C24H27N5O3S; 465.1834.
4.6.9. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-ylthio)-
N-2,3-dimethylphenylacetamide (6i)
4.6.13. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-
ylthio)-N-3,4-dimethylphenylacetamide (6m)
White amorphous powder; Yield: 96%; M.P.: 180–183˚C; IR (KBr)
White amorphous powder; Yield: 92%; M.P.: 164–167˚C; IR (KBr)
–
–
–
–
–
– –
νmax: 3356 (N H), 3034 (Ar-H), 2943 (C H), 1685, 1661 (C O),
νmax: 3350 (N H), 3031 (Ar-H), 2937 (C H), 1681, 1661 (C O),
–
1
1
– –
– –
– –
–
–
1615–1550 (Ar-C C, C N), 1267 (C N); H NMR (400 MHz, CD3OD):
1612–1558 (Ar-C C, C N), 1269 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.64 (2H, m, H-3
α
ʹ,5α
ʹ), 2.02 (2H, m, H-3βʹ,5βʹ), 2.07 (3H, s, CH3), 2.24
δ 1.81 (2H, m, H-3
αʹ,5αʹ), 2.11 (2H, m, H-3βʹ,5βʹ), 2.19 (3H, s, CH3), 2.22
(3H, s, CH3), 3.23 (1H, m, H-4ʹ), 3.04 (2H, m, H-2
αʹ,6α
ʹ), 4.08 (2H, m, H-
(3H, s, CH3), 3.09 (2H, m, H-2αʹ,6αʹ), 3.16 (1H, m, H-4ʹ), 4.11 (2H, s, H-
2βʹ,6βʹ), 4.27 (2H, s, H-2′′′), 6.92 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.00 (1H, d, J
= 7.5 Hz, H-4ʹʹʹʹ), 7.06 (1H, d, J = 7.5 Hz, H-6ʹʹʹʹ), 7.13 (1H, d, J = 7.5 Hz,
H-5ʹʹʹʹ), 7.22 (2H, d, J = 7.3 Hz, H-3ʹʹ,5ʹʹ), 7.45 (2H, d, J = 7.3 Hz, H-
2ʹʹ,6ʹʹ), 13C NMR (100 MHz, CD3OD): δ 20.1 (CH3), 20.7 (CH3), 28.7 (C-
2′′′), 4.13(2H, m, H-2βʹ,6βʹ), 6.99 (1H, t, J = 7.4 Hz, H-4ʹʹ), 7.03 (1H, d, J
= 8.0 Hz, H-6ʹʹʹʹ), 7.22 (1H, d, J = 8.0 Hz, H-5ʹʹʹʹ), 7.25 (2H, t, J = 7.4 Hz,
H-3ʹʹ,5ʹʹ), 7.351 (2H, d, J = 7.4 Hz, H-2ʹʹ,6ʹʹ), 7.36 (1H, s, H-2ʹʹʹʹ); 13C NMR
(100 MHz, CD3OD): δ 19.3 (CH3), 20.0 (CH3), 29.7 (C-3ʹ,5ʹ), 34.2 (C-4ʹ),
12

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
37.2 (C-2ʹʹʹ), 44.1 (C-2ʹ,6ʹ), 118.5 (C-2ʹʹʹʹ), 121.9 (C-2ʹʹ,6ʹʹ), 122.2 (C-6ʹʹʹʹ),
123.9 (C-4ʹʹ), 129.3 (C-3ʹʹ,5ʹʹ), 130.6 (C-5ʹʹʹʹ), 133.6 (C-3ʹʹʹʹ), 133.8 (C-4ʹʹ),
buffer saline, 50 mM, pH 7.4) at 1:1 ratio. An equal volume of
lymphocyte separation medium (density 1.077 g/mL, at 20˚C, Cat. No.
L0560 Lymphosep, Biowest, USA) was added in 15 mL Falcon tube. The
diluted blood was then loaded onto the surface of the medium slowly to
avoid the mixing of blood in the medium. The contents were centrifuged
at 1200 × g for 20 min at 20˚C using a swing rotor. The ring of leukocytes
was collected at the interface of lymphocyte separation medium and
plasma by a Pasteur pipette. Collected cells were washed twice with 10
mL PBS 20˚C at 300 × g. The final pellet obtained was re-suspended in
0.5 mL PBS and the total volume of MNCs was maintained to the
required concentration of 20,000 per 10 µL.
–
137.9 (C-1ʹʹʹʹ), 140.4 (C-4ʹʹ), 157.4 (C O), 165.1 (C-2), 166.5 (C-5),
–
171.1 (C-1′′′); HR-EI-MS (m/z): 465.1854 [M]+ calculated for
C24H27N5O3S; 465.1834.
4.6.14. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-
ylthio)-N-3,5-dimethylphenylacetamide (6n)
White amorphous powder; Yield: 97%; M.P.: 176–177˚C; IR (KBr)
–
–
–
νmax: 3350 (N H), 3031 (Ar-H), 2948 (C H), 1680, 1661 (C O),
–
1
– –
–
1611–1557 (Ar-C C, C N), 1264 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.81 (2H, m, H-3
α
ʹ,5
αʹ), 2,10 (2H, m, H-3βʹ,5βʹ), 2.26 (6H, s, CH3), 3.12
(2H, m, H-2 ʹ,6
α
α
ʹ), 3.18 (1H, m, H-4ʹ), 4.12 (2H, s, H-2′′′), 4.15 (2H, m,
4.9. MTT assay using MNCs
H-2βʹ,6βʹ), 6.75 (1H, s, H-4ʹʹʹʹ), 7.00 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.16 (2H, s,
H-2ʹʹʹʹ,6ʹʹʹʹ), 7.25 (1H, t, J = 7.5 Hz, H-3ʹʹ,5ʹʹ), 7.34 (1H, d, J = 7.5 Hz, H-
2ʹʹ,6ʹʹ); 13C NMR (100 MHz, CD3OD): δ 21.5 (CH3), 29.5 (C-3ʹ,5ʹ), 34.1 (C-
4ʹ), 37.2 (C-2ʹʹʹ), 44.9 (C-2ʹ,6ʹ), 118.7 (C-2ʹʹʹʹ,6ʹʹʹʹ), 121.9 (C-2ʹʹ,6ʹʹ), 123.9
MMT assay was carried out with some modifications as reported
earlier [41,42]. A known volume of PBS (50 mM, pH 7.4) was added per
well followed by the addition of 10 µL test compounds or solvent and 10
µL MNCs in 96 well plate. The contents were incubated for two hours at
(C-4ʹʹ), 127.0 (C-4ʹʹʹʹ), 129.3 (C-3ʹʹ,5ʹʹ), 138.7 (C-1ʹʹʹʹ), 139.4 (C-3ʹʹʹʹ,5ʹʹʹʹ),
′′′
–
–
140.4 (C-1ʹʹ), 157.4 (C O), 165.1 (C-2), 166.6 (C-5), 171.1 (C-1 ); HR-
37˚C. After given time, 10 μL of 0.25 mg/mL MTT solution was added per
EI-MS (m/z): 465.1855[M]+ calculated for C24H327N5O3S; 465.1834.
well. Plate was incubated overnight for 18–20 h in incubator at 37˚C.
After incubation, DMSO (100 L) was added to dissolve the formazon
μ
4.6.15. 2-(5-(1-Phenylcarbamoylpiperdine)-4H-1,3,4-oxadiazole-2-
ylthio)-N-phenylacetamide (6o)
crystals to make a total volume of 200 μL. Absorbance was measured
after two hours at 540 nm. Experiments were carried out in triplicates
along with the controls.
White amorphous powder; Yield: 98%; M.P.:154–156˚C; IR (KBr)
–
–
–
νmax: 3353 (N H), 3037 (Ar-H), 2945 (C H), 1685, 1669 (C O),
–
Cell viability(%) = (Abs. of sam ple ꢀ ꢀ Abs. of blank/Abs. of control
ꢀ ꢀ Abs. of blank) × 100.
1
– –
–
1615–1557 (Ar-C C, C N), 1266 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.81 (2H, m, H-3
αʹ,5αʹ), 2.09 (2H, m, H-3βʹ,5βʹ), 3.08 (2H, m, H-
2
αʹ,6
α
ʹ), 3.15 (1H, m, H-4ʹ), 4.11 (2H, s, H-2′′′), 4.14 (2H, m, H-2βʹ,6βʹ),
6.99 (1H, t, J = 7.6 Hz, H-4ʹʹ), 7.08 (1H, t, J = 7.6 Hz, H-4ʹʹʹʹ), 7.23 (2H, t,
J = 7.6 Hz, H-3ʹʹ,5ʹʹ), 7.28 (2H, t, J = 7.6 Hz, H-3ʹʹʹʹ,5ʹʹʹʹ), 7.32 (2H, d, J =
7.6 Hz, H-2ʹʹ,6ʹʹ), 7.51 (2H, d, J = 7.6 Hz, H-2ʹʹʹʹ,6ʹʹʹʹ); 13C NMR (100 MHz,
CD3OD): δ 29.4 (C-3ʹ,5ʹ), 33.9 (C-4ʹ), 37.1 (C-2ʹʹʹ), 43.9 (C-2ʹ,6ʹ), 120.6
(C-2ʹʹʹʹ,6ʹʹʹʹ), 121.6 (C-2ʹʹ,6ʹʹ), 123.7 (C-4ʹʹʹʹ), 125.2 (C-4ʹʹ), 129.1 (C-3ʹʹ,5ʹʹ),
4.10. Flow cytometry analysis
The cytotoxic behavior of the selected molecules on the MNCs was
carried out by flow cytometric analysis using Annexin V-FITC and pro-
pidium iodide (PI) as reported earlier by our group [35]. The suitable
dilutions of the compounds were made, that is, at the concentrations of
their IC50 values. The two different concentrations (IC50 value and 3 ×
IC50 values) of each selected drug were incubated with MNCs for 45 min.
The negative control was also taken for comparison. The cells (1 × 106
cells) were transferred to FACS tubes. The Annexin V-FITC (10 µL) was
added to the tubes followed by the addition of 5 µL PI and contents were
vortexed for few seconds. Then cells were incubated at room tempera-
ture for 15 min and Annexin V binding buffer (400 µL) was added. The
stained cells were analyzed on BD FAC scan (USA) using flow cytometer.
The data was analyzed using the BD FAC scan software and through
Microsoft excel. The experiments were performed in triplicate.
–
129.4 (C-3ʹʹʹʹ,5ʹʹʹʹ), 138.5 (C-1ʹʹʹʹ), 140.0 (C-1ʹʹ), 157.3 (C O), 164.9 (C-2),
–
166.3 (C-5), 170.7 (C-1′′′); HR-EI-MS (m/z): 437.1541 [M]+ calculated
for C22H23N5O3S; 437.1521.
4.7. 15-LOX inhibition assay
The assay was performed according to the chemiluminescence
method of Kondo and coworkers [40] with some minor modifications as
reported earlier [41]. Briefly, a total volume of 100 µL of reaction
mixture contained 60 µL of borate buffer (200 mM, pH 9.0), 10 µL of test
compound or solvent, and 10 µL soybean 15-LOX enzyme. This mixture
was incubated at 25˚C in dark for 5 min. Then 10 µL solution of luminol
(3 nM) containing cytochrome c (1 nM) was added to each well and
luminescence was measured by BioTek HTX 96-well plate reader in
luminescence mode. The reaction was initiated by the addition of 10 µL
substrate solution. Chemiluminescence of the reaction contents was
measured for 100 to 300 sec. All experiments were performed in tripli-
cates. Quercetin was used as a positive control. The percentage of in-
hibition was calculated using the following formula:
4.11. In silico studies
4.11.1. Protein structure finding and ligand formation
The initial procedure for the study includes the retrieval of the
protein structure from protein data bank against the name 5-LOX and
15-LOX. The retrieved protein crystal structure from protein data bank
with resolution 2.0 to 2.5 Å were 5-LOX and 15-LOX human with PDB id:
6N2W and PDB id: 4NRE while for the soybean with PDB id: 1RRH
[7,43,44]. All the synthesized compounds were taken as ligand and were
drawn using Chemsketch [45]. The ligands were then optimized using
Biovia Discovery Studio 2016 [46]. Quercetin was used as standard
against lipoxygenase enzyme, taken from Pubchem with CID: 5,280,343
[47].
Inhibition(%) = (Abs of control ꢀ ꢀ Abs of test com p/Abs of control) × 100
The active compounds were suitably diluted and their percentage in-
hibitions were determined. The obtained data was used for the
computation of IC50 values using Ez–Fit Enzyme kinetics software
(Perrella Scientific Inc. Amherst, USA).
4.11.2. Docking
4.8. Cell viability assays
The molecular docking is referred as the main procedure for identi-
fying the binding of different compounds against the enzyme using the
basic idea of enzyme-substrate complex. The molecular docking was
performed using Autodock and Autodock Vina softwares and later
visualized in Discovery Studio [48,49]. The heteroatoms and water
molecules from the downloaded enzymes were removed using Biovia
4.8.1. Isolation of MNCs
MNCs were isolated and purified as reported earlier by our group
[35]. Blood (3 mL) was collected in an EDTA tube from the healthy
volunteer donor with consent, which was diluted with PBS (phosphate
13

B. Bashir et al.
Bioorganic Chemistry 115 (2021) 105243
their correlation with histopathology of liver, Int. J. Pharm. Edu. Res. 51 (2017)
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Discovery Studio 2016. The partial charges in the enzymes while the
polar hydrogens in the ligands were added using Autodock.
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[22] F. Naeem, M. Ansar, K. Hameed, S. Ayub, K. Haq, I. Tahir, M.N. Mahmood,
Synthesis, structural studies and biological activities of three new 2-
(pentadecylthio)-5-aryl-1,3,4-oxadiazole, Act. Pharm. Sci. 55 (2017) 50–59.
[23] S. Triloknadh, C.V. Rao, B. Nagaraju, H. Balaji, M. Balaji, Design and synthesis of
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one derivatives, Bioorg. Med. Chem. 13 (2005) 3797–3809.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Thanks are due to the Higher Education Commission (HEC) of
Pakistan for funding the NRPU Project No. 4950 to M.A. and research
assistantship to W. Shahid. Prof N. Riaz is thankful to Prof. Dr. Harald
Gross, Institute of Pharmaceutical Biology, University of Tuebingen,
Tuebingen, Germany for providing NMR and Mass analysis facility.
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